ABSTRACT We know that the gut microbiome functions as an accessory organ system that plays critical roles in human digestion; the production of essential vitamins, neurotransmitters, and other small molecules; and in the regulation of the immune system. We also know that changes in the gut microbiota can lead to disease, especially inflammatory bowel disease and related inflammatory disorders. However, we know very little about the molecules and mechanisms that connect gut microbiota to these diseases. As the bacteria in the gut microbiota sense and respond to their environment with small molecules, it's likely that some of these molecules are key regulators of both pro-inflammatory and anti-inflammatory responses. This project seeks to identify these molecules and the mechanisms by which they operate. The project stems from a rich data set (DIABIMMUNE) that revealed a number of robust correlations between the appearance, or disappearance, of members of the gut microbiota and the development of dysregulated immune responses and disease in infants with genetic pre-disposition to type 1 diabetes. We will focus on ~40 strains that appear to play outsize roles in disease progression to discover what pro- or anti-inflammatory small molecule signals they produce. In Specific Aim 1 we will culture patient-derived strains of these bacteria under a variety of conditions, capture the small molecules produced, and measure their ability to regulate a pro-inflammatory signal (TNFalpha) and an anti- inflammatory signal (IL-10). The most potent and selective regulators will advance to Specific Aim 2 in which bioassay-guided fractionation will guide the isolation of active molecules, which will then be structurally characterized with spectroscopic (both nuclear magnetic resonance and mass spectrometry) and imaging (X-ray diffraction) techniques. With known molecules linked to confirmed biological activity in hand, we will prioritize hits based on their presence in the DIABIMMUNE stool samples, which we have archived. Only bioactive molecules present in human samples will be analyzed in specific Aim 3 to determine mechanism. Because the signaling pathways through which these molecules exert their biological effects are complex, we will employ a specially designed and newly implemented assay that simultaneously measures more than 50 transcription factors that regulate inflammation, immunity, metabolism and cell stress. By profiling transcription factor activity and comparing to known compounds, we will be able to classify molecules based on their activity profiles. We will further characterize their mechanism of action using a variety of functional assays on both patient derived myeloid cells as well as colonic and ileal mucosa cell lines that develop into four distinct cell types that form three-dimensional structures including crypts covered by a secreted mucus layer. At the end of the pipeline formed by these three specific aims, small molecules that regulate immune responses along annotated pathways will be known, and this identification of both molecule and mechanism will provide insights into both basic biology and potential therapeutic interventions.